Development of validated analytical methods for selected Cardiovascular Drugs in formulations and its application in in-vitro interaction studies.

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DEVELOPMENT OF VALIDATED ANALYTICAL METHODS

FOR SELECTED CARDIOVASCULAR DRUGS IN

FORMULATIONS AND ITS APPLICATION IN IN-VITRO

INTERACTION STUDIES

Dissertation submitted to

THE TAMIL NADU DR. M. G. R. MEDICAL UNIVERSITY,

CHENNAI

For the award of Degree of

DOCTOR OF PHILOSOPHY

In

PHARMACY

Submitted by

V. Shivashankar M.Pharm.,

Under the Guidance of

Dr. T.K. Ravi M.Pharm., PhD., FAGE

DECEMBER 2015

COLLEGE OF PHARMACY

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S.No Title Page No LIST OF ABBREVIATIONS

LIST OF TABLES LIST OF FIGURES

1 INTRODUCTION 1

2 AIM AND OBJECTIVES 5

3 LITERATURE REVIEW 6

4 SCOPE AND PLAN OF WORK 10

5 MATERIALS AND INSTRUMENTS 12

6 EXPERIMENTAL METHODS

6.1.1 Development of validated UV spectrophotometric method for the determination of dronedarone hydrochloride in tablet dosage form

17

6.1.2 Development of validated spectrofluorimetric method for the determination of dronedarone hydrochloride in tablet dosage form

19

6.1.3 Development of validated HPTLC method for the determination of dronedarone hydrochloride in tablet dosage form

22

6.1.4 Development of validated stability indicating RP-HPLC method for the determination of dronedarone hydrochloride in tablet dosage form

25

6.2.1 Development of validated UV spectrophotometric method for the determination of levosimendan in injection

32

6.2.2 Development of validated spectrofluorimetric method for the determination of levosimendan in injections

34

6.2.3 Development of validated HPTLC method for the determination of levosimendan in injection

37

6.2.4 Development of validated stability indicating RP-HPLC method for the determination of levosimendan in injection dosage form

40

6.3.1 Development of validated UV spectrophotometric method for the determination of rivaroxaban in tablet dosage form

46

6.3.2 Development of validated spectrofluorimetric method for the determination of rivaroxaban in tablet dosage form

48

6.3.3 Development of validated stability indicating HPTLC method for the determination of rivaroxaban in tablet dosage form

51

6.3.4 Development of validated RP-HPLC method for the determination of rivaroxaban in tablet dosage form

55

6.4.1 Development of validated UV spectrophotometric method for the determination of ticagrelor in tablet dosage form

60

6.4.2 Development of validated HPTLC method for the determination of ticagrelor in tablet dosage form

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6.4.3 Development of validated stability indicating RP-HPLC method for the determination of ticagrelor in tablet dosage form

65

7 RESULTS & ANALYSIS

7.1.1 Development of validated UV spectrophotometric method for the determination of dronedarone hydrochloride in tablet dosage form

69

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122

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179

8 DISCUSSION

8.1.1 Development of validated UV spectrophotometric method for the determination of dronedarone hydrochloride in tablet

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210

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9 SUMMARY & CONCLUSION

9.1 Development of validated analytical methods for the determination of dronedarone hydrochloride

218

9.2 Development of validated analytical methods for the determination of ticagrelor

223

9.3 Development of validated analytical methods for the determination of rivaroxaban

227

9.4 Development of validated analytical methods for the determination of ticagrelor

231

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LIST OF ABBREVIATIONS

ACN - Acetonitrile

ANOVA - Analysis of Variance

BSA - Bovoine Serum Albumin

CAS - Cerric Ammonium Sulphate

CHF - Congestive Heart Failure

CVD - Cardiovascular Diseases

DDI - Drug-Drug Interactions

DMSO - Dimethyl Sulphoxide

gm - Gram

HPLC - High Performance Liquid Chromatography

HPTLC - High Performance Thin Layer Chromatography

H2O2 - Hydrogen peroxide

hrs - Hours

ICH - International Conference on Harmonization

KH2PO4 - Potassium dihydrogen orthophosphate

LOD - Limit of detection

LOQ - Limit of quantification

M - Molar

mg - Milligram

min - Minute

ml - Millilitre

mM - Millimolar

NaOH - Sodium Hydroxide

NaCl - Sodium Chloride

ng - Nanogram

nm - Nanometer

OPA - Ortho Phosphoric Acid

PDA - Photo Diode Array

Rf - Retardation factor

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RSD - Relative standard deviation

Rt - Retention time

SD - Standard Deviation

TEA - Triethylamine

TFA - Trifluro acetic acid

Tf - Tailing factor

λmax - Wavelength of maximum absorbance

μg - Microgram

μL - Microlitre

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LIST OF TABLES

Table No. Title

Development of validated UV spectrophotometric method for the determination of dronedarone hydrochloride in tablet dosage form

1 Calibration data for dronedarone hydrochloride by second derivative UV spectrophotometric method

2 Recovery data of dronedarone hydrochloride

3 Precision data of dronedarone hydrochloride

4 Stability data of Dronedarone Hydrochloride (bench top)

5 Stability data of Dronedarone Hydrochloride (refrigerated)

6 Optical characteristics of dronedarone hydrochloride

7 Assay of dronedarone hydrochloride tablets

Development of validated spectrofluorimetric method for the determination of dronedarone hydrochloride in tablet dosage form

8 Calibration data for dronedarone hydrochloride by spectrofluorimetric method

11 Stability data of dronedarone hydrochloride (bench top

12 Stability data of dronedarone hydrochloride (refrigerated)

13 Assay of dronedarone hydrochloride tablets by spectrofluorimetry

Development and validation of HPTLC method for

determination of dronedarone hydrochloride in tablet dosage form

14 Linearity data for dronedarone hydrochloride by HPTLC method

16 Repeatability data of sample application of dronedarone hydrochloride

17 Repeatability data of sample measurement data of dronedarone hydrochloride

19 Stability data of dronedarone hydrochloride (bench top)

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Development and validation of stability indicating RP-HPLC method for determination of dronedarone hydrochloride in tablet dosage form

22 Linearity data for dronedarone hydrochloride

24 Repeatability data of dronedarone hydrochloride

26 Stability data of dronedarone hydrochloride (bench top)

28 System suitability parameters of dronedarone hydrochloride

29 Assay results of dronedarone hydrochloride tablets by HPLC

30 Forced degradation results of dronedarone hydrochloride

31 Results of protein binding study of dronedarone hydrochloride

32 Results of in-vitro_{interaction study of dronedarone hydrochloride} with aspirin

33 Results of in-vitro_{interaction study of dronedarone hydrochloride} with atorvastatin

Development of validated UV spectrophotometric method for the determination of levosimendan in injection form

34 Calibration data for levosimendan by UV spectrophotometric method

35 Recovery data of levosimendan by UV method

36 Precision data of levosimendan

37 Stability data of levosimendan (bench top stored stock)

38 Stability data of levosimendan (refrigerated stock)

39 Optical characteristics of levosimendan

40 Assay results of levosimendan injection

Development of validated spectrofluorimetric method for the determination of levosimendan injections

41 Calibration data for levosimendan by spectrofluorimetric method

42 Recovery data of levosimendan

44 Stability data of levosimendan (bench top)

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46 Assay results of levosimendan injection by spectrofluorimetry

determination of levosimendan in injections

47 Linearity data for levosimendan by HPTLC method

49 Repeatability data of sample application of levosimendan

50 Repeatability data of sample measurement of levosimendan

52 Stability data of Levosimendan (bench top)

53 Stability data of Levosimendan (Refrigerated)

54 Assay results of levosimendan injections by HPTLC method

Development and validation of stability indicating RP-HPLC method for determination of levosimendan in injection form

55 Linearity data for levosimendan

57 Repeatability data of levosimendan

59 Stability data of levosimendan (bench top)

60 Stability data of levosimendan (refrigerated)

61 System suitability parameters of levosimendan

62 Assay of levosimendan injections

63 Forced degradation results of levosimendan

64 Protein binding of levosimendan

65 Displacement of levosimendan from protein by aspirin

66 Displacement of levosimendan from protein by clopidogrel

67 Displacement of levosimendan from protein by atorvastatin

Development of validated UV spectrophotometric method for the determination of rivaroxaban in tablet dosage form

68 Calibration data of rivaroxaban

69 Recovery data of rivaroxaban

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71 Stability data of rivaroxaban (bench top)

72 Stability data of rivaroxaban (refrigerated)

73 Optical characteristics of rivaroxaban

74 Assay of rivaroxaban tablets by UV method

Development of validated spectrofluorimetric method for the determination of rivaroxaban in tablet dosage form

75 Calibration data for rivaroxaban by spectrofluorimetric method

77 Precision data of rivaroxaban

80 Assay of rivaroxaban Tablets

Development and validation of stability indicating HPTLC method for determination of rivaroxaban in tablet dosage form

81 Linearity data for rivaroxaban

82 Recovery data of rivaroxaban by HPTLC method

83 Repeatability of sample application

84 Repeatability of sample measurement

88 Assay of rivaroxaban tablets by HPTLC method

89 Forced degradation results of rivaroxaban

Development and validation of RP-HPLC method for determination of rivaroxaban in tablet dosage form

90 Linearity data for rivaroxaban

92 Repeatability data of rivaroxaban

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97 Assay of rivaroxaban tablets

98 Protein binding of rivaroxaban

99 Displacement of rivaroxaban from protein by aspirin

100 Displacement of rivaroxaban from protein by clopidogrel

Development of validated UV spectrophotometric method for the determination of ticagrelor in tablet dosage form

101 Calibration data of ticagrelor

102 Recovery data of ticagrelor

103 Precision data of ticagrelor

104 Stability data of ticagrelor (bench top)

105 Stability data of ticagrelor (refrigerated)

106 Optical characteristics of ticagrelor

107 Assay of ticagrelor tablets

determination of ticagrelor in tablet dosage form

108 Linearity data for ticagrelor

110 Repeatability of sample application

111 Repeatability of sample measurement

114 Stability data of ticagrelor (refrigerated)

115 Assay of ticagrelor tablets by HPTLC

Development and validation of stability indicating RP-HPLC method for determination of ticagrelor in tablet dosage form

116 Linearity data for ticagrelor

118 Repeatability data of ticagrelor

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123 Assay of ticagrelor tablets by HPLC

124 Forced degradation results of ticagrelor

125 Comparison of developed analytical methods for the determination of dronedarone hydrochloride

126 Statistical comparison of the percentage assay of dronedarone hydrochloride tablets determined by developed analytical methods 127 Comparison of UV spectroscopic method developed with reported

methods of determination of dronedarone hydrochloride

128 Comparison of HPTLC method developed with reported methods of determination of dronedarone hydrochloride

129 Comparison of HPLC method developed with reported methods of determination of dronedarone hydrochloride

130 Statistical comparison of the percentage protein binding of dronedarone hydrochloride in presence of interacting drugs

131 Comparison of developed analytical methods for the determination of levosimendan

132 Statistical comparison of the percentage assay of levosimendan injections determined by developed analytical methods

133 Statistical comparison of the percentage protein binding of levosimendan in presence of interacting drugs

134 Comparison of developed analytical methods for the determination of rivaroxaban

135 Statistical comparison of the percentage assay of rivaroxaban tablets determined by developed analytical methods

136 Comparison of UV spectroscopic method developed with reported methods of determination of rivaroxaban

137 Comparison of HPTLC method developed with reported methods of determination of rivaroxaban

138 Comparison of HPLC method developed with reported methods of determination of rivaroxaban

139 Statistical comparison of the percentage protein binding of rivaroxaban in presence of interacting drugs

140 Comparison of developed analytical methods for the determination of ticagrelor

141 Statistical comparison of the percentage assay of ticagrelor tablets determined by developed analytical methods

142 Comparison of UV spectroscopic method developed with reported methods of determination of ticagrelor

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LIST OF FIGURES

Figure No. Title

Development of validated UV spectrophotometric method for the determination of dronedarone hydrochloride in tablet dosage form

1 Zero order spectrum of dronedarone hydrochloride

2 First order derivative spectrum of dronedarone hydrochloride

3 Second order derivative spectra of dronedarone hydrochloride

4 Second order derivative overlay spectra of dronedarone hydrochloride

5 Calibration graph of dronedarone hydrochloride

6 Second order derivative of dronedarone hydrochloride tablets

Development of validated spectrofluorimetric method for the determination of dronedarone hydrochloride in tablet dosage form

7 Excitation spectra of Cerrium (III) (formed after oxidation of dronedarone hydrochloride)

8 Emission spectra of Cerrium (III) (formed after oxidation of dronedarone hydrochloride)

9 Overlay emission spectra of Cerrium (III) (formed after oxidation of dronedarone hydrochloride)

10 Linearity graph of dronedarone hydrochloride

11 Emission spectra of Cerrium (III) (formed after oxidation of dronedarone hydrochloride tablets)

determination of dronedarone hydrochloride in tablet dosage form

12-16 Chromatograms of Standards of Dronedarone Hydrochloride obtained by HPTLC method

18 Chromatogram of dronedarone hydrochloride in tablets

Development and validation of stability indicating RP-HPLC method for determination of dronedarone hydrochloride in tablet dosage form

19 Standard chromatogram of dronedarone hydrochloride

21-25 Chromatograms of standards of Dronedarone Hydrochloride

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27 UV-spectrum of dronedarone hydrochloride

28 Chromatogram of dronedarone hydrochloride at 0 hrs of acid hydrolysis

29 Spectrum of dronedarone hydrochloride peak after acid hydrolysis at 0 time

30 Chromatogram of dronedarone hydrochloride after 2 hrs of acid hydrolysis

31 Spectrum of dronedarone hydrochloride peak after acid hydrolysis at 2 hrs

32 Chromatogram of dronedarone hydrochloride at 0 hrs of base hydrolysis at 0 hrs

33 Spectrum of dronedarone hydrochloride peak after base hydrolysis at 0 time

34 Chromatogram of dronedarone hydrochloride after 2 hrs of base hydrolysis

35 Spectrum of dronedarone hydrochloride peak after base hydrolysis at 2 hrs

36 Chromatogram of dronedarone hydrochloride at 0 hrs of neutral hydrolysis

37 Spectrum of dronedarone hydrochloride peak after neutral hydrolysis at 0 time

38 Chromatogram of dronedarone hydrochloride after 2 hrs of neutral hydrolysis

39 Spectrum of dronedarone hydrochloride peak after neutral hydrolysis at 2 hrs

40 Chromatogram of dronedarone hydrochloride at 0 hrs of oxidative degradation

41 Spectrum of dronedarone hydrochloride peak after oxidative degradation at 0 time

42 Chromatogram of dronedarone hydrochloride after 2 hrs of oxidative degradation

43 Spectrum of dronedarone hydrochloride peak after oxidative degradation at 2 hrs

44 Chromatogram of dronedarone hydrochloride after 6 hrs of photolytic degradation

45 Spectrum of dronedarone hydrochloride peak after photolytic degradation at 6 hrs

46 Chromatogram of simultaneous elution dronedarone hydrochloride with aspirin

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48 Chromatogram of unbound dronedarone hydrochloride after in-vitro displacement interaction with aspirin

49 Chromatogram of unbound dronedarone hydrochloride after in-vitro displacement interaction with atorvastatin

Development of validated UV spectrophotometric method for the determination of levosimendan in injection form

50 Standard UV spectrum of levosimendan

51 Overlay UV spectrum of levosimendan standards (1 µg/ml -5 µg/ml)

52 Linearity graph of levosimendan

53 UV Spectrum of levosimendan formulation

Development of validated spectrofluorimetric method for the determination of levosimendan injections

54 Excitation spectra of cerium (III) (formed after oxidation of levosimendan)

55 Emission spectra of cerium (III) (formed after oxidation of levosimendan)

56 Overlay emission spectra of cerium (III) (formed after oxidation of levosimendan)

58 Emission spectra of cerium (III) (formed after oxidation of levosimendan injection)

determination of levosimendan in injections

60-64 Chromatograms of standards of levosimendan obtained by HPTLC

65 Chromatogram of levosimendan injection

Development and validation of stability indicating RP-HPLC method for determination of levosimendan in injection form

66 Standard chromatogram of levosimendan by HPLC method

68-73 Chromatograms of standards of levosimendan obtained by HPLC method

74 Chromatogram of levosimendan injection

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76 Chromatogram of levosimendan at 0 hrs of acid hydrolysis

77 Spectrum of levosimendan peak at 0 time of acid hydrolysis

78 Chromatogram of levosimendan after 2 hrs of acid hydrolysis

79 Spectrum of levosimendan peak 2 hrs of acid hydrolysis

80 Chromatogram of levosimendan at 0 hrs of base hydrolysis

81 Spectrum of levosimendan peak at 0 time of base hydrolysis

82 Chromatogram of levosimendan after 2 hrs of base hydrolysis

83 Spectrum of levosimendan peak after 2 hrs of base hydrolysis

84 Chromatogram of levosimendan at 0 hrs of neural hydrolysis

85 Spectrum of levosimendan peak at 0 time of neutral hydrolysis

86 Chromatogram of levosimendan after 2hrs of neutral hydrolysis

87 Spectrum of levosimendan peak after 2 hrs of neutral hydrolysis

88 Neutral hydrolysis - spectrum of degradant peak at 4.8min

89 Neutral hydrolysis - spectrum of degradant peak at 8.6min

90 Chromatogram of levosimendan at 0 hrs of oxidative degradation

91 Spectrum of levosimendan peak at 0 time of oxidative degradation

92 Chromatogram of levosimendan after 2 hrs of oxidative degradation

93 Oxidative stress- spectrum of degradant peak at 4.8 min

94 Chromatogram of levosimendan after 4 hrs of photolytic degradation

95 Spectrum of levosimendan peak after 4 hrs Photolytic stress

96 Chromatogram of simultaneous elution of levosimendan with aspirin

97 Chromatogram of simultaneous elution of levosimendan with clopidogrel

98 Chromatogram of simultaneous elution of levosimendan with atorvastatin

99 Chromatogram of unbound levosimendan after equilibrium dialysis with protein

100 Chromatogram of levosimendan unbound levosimendan after interaction with aspirin

101 Chromatogram of levosimendan unbound levosimendan after interaction with clopidogrel

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Development of validated UV spectrophotometric method for the determination of rivaroxaban in tablet dosage form

103 UV-spectrum of Rivaroxaban standard

104 Overlay spectrum of rivaroxaban standards (5-25 µg/ml)

105 Calibration graph of rivaroxaban

106 Spectrum of rivaroxaban tablets

Development of validated spectrofluorimetric method for the determination of rivaroxaban in tablet dosage form

107 Excitation spectra of cerium (III) (formed after oxidation of rivaroxaban)

108 Emission spectra of cerium (III) (formed after oxidation of rivaroxaban)

109 Overlay emission spectra of cerium (III) (formed after oxidation of rivaroxaban)

110 Linearity graph of rivaroxaban

111 Emission spectra of cerium (III) (formed after oxidation of rivaroxaban tablets)

Development and validation of stability indicating HPTLC method for determination of rivaroxaban in tablet dosage form

112-116 Chromatograms of Standards of rivaroxaban

117 Linearity graph of rivaroxaban by HPTLC

118 Chromatogram of rivaroxaban tablets

119 Chromatogram of acid hydrolyzed rivaroxaban at 0 hr

120 Chromatogram of acid hydrolyzed rivaroxaban after 2 hrs

121 Chromatogram of base hydrolyzed rivaroxabanat 0 hr

122 Chromatogram of base hydrolyzed rivaroxabanafter 2 hrs

123 Chromatogram of neutral hydrolyzed rivaroxabanat 0 hr

124 Chromatogram of neutral hydrolyzed rivaroxabanafter 2 hrs

125 Chromatogram of oxidative stressed rivaroxaban at 0 hr

126 Chromatogram of oxidative stressed rivaroxaban after 2 hrs

127 Chromatogram of rivaroxaban after 6 hrs of photolytic degradation

Development and validation of RP-HPLC method for determination of rivaroxaban in tablet dosage form

128 Standard chromatogram of rivaroxaban

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130-134 Chromatograms of Standards of rivaroxaban

135 Chromatogram of rivaroxaban formulation

136 Chromatogram of simultaneous elution rivaroxaban with aspirin

137 Chromatogram of simultaneous elution rivaroxaban with clopidogrel

138 Chromatogram of unbound rivaroxaban after equilibrium dialysis

139 Chromatogram of unbound rivaroxaban after in-vitro interaction with aspirin

140 Chromatogram of unbound rivaroxaban after in-vitro interaction with

Development of validated UV spectrophotometric method for the determination of ticagrelor in tablet dosage form

141 UV-spectrum of standard ticagrelor

142 Overlay spectrum of ticagrelor standards (0.2-1.0 µg/ml)

143 Calibration graph of ticagrelor

144 UV- spectrum of ticagrelor tablets

determination of ticagrelor in tablet dosage form

145-149 Chromatograms of Standards of ticagrelor

150 Linearity graph of ticagrelor

151 Chromatogram of ticagrelor tablets

Development and validation of stability indicating RP-HPLC method for determination of ticagrelor in tablet dosage form

152 Standard chromatogram of ticagrelor

153 Linearity graph of ticagrelor

154-159 Chromatograms of Standards of ticagrelor

160 HPLC Chromatogram of ticagrelor tablets

161 UV spectrum of ticagrelor

162 Chromatogram of ticagrelor at 0 hrs of acid hydrolysis

163 Spectrum of ticagrelorpeak at 0 time of acid hydrolysis

164 Chromatogram of ticagrelor after 2 hrs of acid hydrolysis

165 Spectrum of ticagrelorpeak after 2 hrs of acid hydrolysis

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167 Chromatogram of ticagrelor at 0 hrs of base hydrolysis

168 Spectrum of ticagrelor peak at 0 time of base hydrolysis

169 Chromatogram of ticagrelor after 2 hrs of base hydrolysis

170 Spectrum of ticagrelor peak after 2 hrs of base hydrolysis

171 Chromatogram of ticagrelor at 0 hrs of neutral hydrolysis

172 Spectrum of ticagrelor peak at 0 time of neutral hydrolysis

173 Chromatogram of ticagrelor after 2 hrs of neutral hydrolysis

174 Spectrum of ticagrelorpeak after 2 hrs of neutral hydrolysis

175 Chromatogram of ticagrelor at 0 hrs of oxidative degradation

176 Spectrum of ticagrelor peak at 0 time of oxidative degradation

177 Chromatogram of ticagrelorafter 2 hrs of o_{xidative degradation}

178 Spectrum of ticagrelorpeak after 2 hrs of o_{xidative degradation}

179 Photolytic degradation after 4 hrs

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1. INTRODUCTION

Drugs are the natural or synthetic chemical compounds which are indented

for pharmacotherapy, diagnosis or prevention of diseases. New drugs are

approved every year and are often used concomitantly with other drugs.

Pharmacotherapy is a complex process, thus appropriate selection of drug is more

challenging. Concomitant use of drugs may result in mild, moderate or severe

drug-drug interactions (DDI).Globally DDI’s have received greater attention from

various statutory bodies1. The new interactions between medications are

increasingly reported. The incidence of DDI-related adverse drug reactions was

found to be 6.5% and most of the events presented important clinical

consequences2. DDIs may results in emergency admission, increased financial

burden to patient and even results in death3. Clinically significant interaction

sometimes causes serious adverse reactions therefore a systematic knowledge of

drug interaction may help to prevent adverse effects4.

DDI’s might result termination of drug development, disapproval for

marketing and withdrwal of drugs from market. Therefore drug interactions

should be considered from the perspective of both investigational drug and

approved drugs which are expected to be used concomitantly with other drugs.

Prospective studies are needed to determine result of major drug interactions5.

Pharmacokinetics drug interactions occur when one drug alters the concentration

of another drug by altering absorption, distribution, bioavailability, metabolism

and excretion.

Distribution interactions involve interactions in protein binding of drugs.

Two major factors affecting protein binding include the presence of other drugs

and the effects of disease. Drug displacement may be clinically significant in case

of potent drugs. Displacement interactions may require icresed dose of a drug or

decreased dose depending on the pharmacokinetics of drug. Knowledge of altered

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In order to predict clinically significant drug interaction that is suspected

to be resulted from protein binding displacement, it is important to validate the

findings using specific in-vitro protein binding studies6. In-vitro DDI studies requires specific analytical techniques with great sensitivities for accurate

estimation of drugs. So it is essential to establish the validated methods for the assessment of in-vitro drug interaction studies.

The analytical technique frequently employed for the estimation of drugs,

and drug combinations is high performance liquid chromatography (HPLC). The

HPLC method offer a multitude of advantages and it is suitable method for

quantitative analysis. The excellent sensitivity makes HPLC suitable for

estimation of drugs in biological fluids7. Advantages of using HPLC include

speed, automation and compatibility8. Thus HPLC method can be effectively

employed in the in-vitro interaction studies.

High Performance Thin Layer Chromatography (HPTLC) is well

established method due to its reliability and accuracy. The advantage of this

method is ability to run multiple samples at same time. It is economical due to

small quantity of mobile phase utilization9.

UV spectroscopy is routinely used analytical method. It is very simple and

does not involve laborious procedures10. Due to the simplicity, accuracy and

reliability UV- spectroscopic method is very useful in quantification of drug and

formulations. Derivative spectrophotometry offers enhanced resolution and bandwidth discrimination by converting normal spectra into first and second order

derivative spectrum.

Spectrofluorimetry method is commonly utilized in quantitative analysis of

drugs and metabolites due to its relatively low cost and greater sensitivity. This

method can be inherently applicable to lower concentration ranges11. The non

fluorogenic compounds can also be estimated by this method by either converting

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Validation ensures quality and reliability of analytical techniques. It is a

process that demonstrate the established techniques are suitable and proved to be

exactly correlating with appropriate standards which is directed for their intended

use. It is necessary to validate the analytical method(s) as per ICH guidelines and

to provide proper and appropriate validation information12. The importance of validation is producing reliable, repeatable and accurate results for routine

analysis. The validation of analytical methods builds high degree of confidence

and reliability on developed method.

Development of stability indicating analytical method measure active

ingredients, that is clearly free from process impurities and degradation

products13. The current guidelines and regulatory requirements mandates

development of analytical methods which posses greater selectivity, sensitivity,

accurate, precise and robust14. Force degradation is essential for development of

stability indicating analytical method. Force degradation studies helps to

determine the degradation pathway and degradation products of the APIs that is probably obtained during storage and facilitate formulation development,

manufacturing and packaging15.

The incidence of cardiovascular diseases (CVDs) has increased and

globally it is one of the leading causes of death. The cardiovascular drugs are the

most widely prescribed drugs and it ranks second in the overall sales of drug in

India16. Cardiovascular drugs are one of the drug categories frequently involved in

drug related problems. Patients taking CVD drugs are more prone to potential

DDI and the factors related to DDI include age, polypharmacy and changes in

pharmacokinetics of drugs. Most of the elderly population is taking cardiovascular

medications and the vulnerability of this population to DDI’s is enormous17. The

potential DDI might results due to the narrow therapeutics index of drugs and

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The DDI potential varies with patient ability to metabolize and excerete

the drug. The DDI potential also depends on duration of concomitant

administration of drugs18. It is essential to determine protein binding in order to

minimize the interaction of coadministered drugs. In order to determine

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2. AIM AND OBJECTIVES

The current study aims on development of validated analytical methods for the selected cardiovascular drugs and its formulation. The cardiovascular drugs selected presently are dronedarone hydrochloride, rivaroxaban, levosimendan and

ticagrelor. These drugs are approved by CDSCO for the different indications to treat of cardiovascular diseases.

The objective of the study was development of simple and sensitive methods for determination of selected cardiovascular drugs in its formulations and application of most sensitive method for in-viro interaction studies. The specific objectives of the study are:

 To develop UV spectrophotometric methods for the determination of dronedarone hydrochloride, rivaroxaban, levosimendan and ticagrelor.

 To develop new spectroflurimetric methods for the estimation of

dronedarone hydrochloride, rivaroxaban and levosimendan.

 To develop RP-HPLC and HPTLC methods for the determination of

dronedarone hydrochloride, rivaroxaban, levosimendan and ticagrelor.  To apply validated analytical methods for the estimation of selected drugs

in formulations.

 To establish the inherent stability characteristics of the selected drugs and

formulations through stress studies as per ICH recommendations.

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3. LITERATURE REVIEW

3.1. Dronedarone Hydrochloride

Mrinalini C. D., et al (2015)19 has reported a validated stability indicating

UV spectrometric method and HPTLC method for determination of dronedarone

hydrochloride. Methanol was used as solvent and absorption maximum was observed at 288nm. The linear range of dronedarone was reported in range of 10-50µg/ml in UV method and 40-200 ng/band by HPTLC. The study compared the results of UV method and HPTLC method and concluded that UV method is more

sensitive.

Pravalika K., et al (2013)20 conducted a study on spectrophotometric

estimation of dronedarone hydrochloride in formulation. Dronedarone shows maximum wavelength at 288 nm using methanol as a solvent. The linearity was

reported in range of 10-35µg/ml.

A study on spectrophotometric estimation of dronedarone was carried out

by Arpan Patel., et al (2012)21. The analytical wavelength of 290 nm was applied

for the estimation of dronedarone. The linear range of 2-30 µg/ml was reported.

Rajyalakshmi C., et al (2013)22 carried out degradation study of

dronedarone by HPLC-UV method. A C18 column was used with mobile phase of ACN and phosphate buffer. The reported LOD was 0.018 µg/ml and LOQ was 0.06 µg/ml with a linearity range of 10-50 µg/ml.

A HPLC method for quantification of dronedarone was demostrated by

Emanual M. P., et al (2013)23. The reported method has linearity in the range of

5-15µg/ml of dronedarone and retention time of 4.7min.

Kishore K., et al (2014)24 performed a study for determination of

dronedarone by RP-HPLC technique. The λmax of the dronedarone was found to be 290 nm. The mobile phase of ACN: water was used in 70:30 ratio. The linearity of dronedarone was observed between 5-35µg/ml.

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column. The ACN and buffer mixture were used as a mobile phase. Linear response was reported between 25-100 µg/ml.

Arpan P., et al (2012)26 has reported a HPLC method for determination of

dronedarone hydrochloride. The Hypersil C18 column with mobile phase comprised of buffer: ACN was used. The reported linearity range was 10-150 µg/ml.

A study on HPTLC method for estimation of dronedarone hydrochloride was carried out by Batuk D., et al (2012)27. Chromatographic separation was

carried out on silicagel G60F254 plates using acetone and methanol as mobile phases. The linear was response found in the concentration range of 200-800ng/band.

3.2. Levosimendan

The enantiomeric separation of levosimendan was described by Rambabu

C. et al (2013)28. The mobile phase consisting of Methyl-tert-butyl ether: ethanol:

trifluoro acetic acid was used. The elution times of S-Levosimendan and

R-Levosimendan were found to be 6.8 min and 11.0 minutes respectively

3.3. Rivaroxaban

Kasad P.A., et al (2013)29 carried out a study on determination of

rivaroxaban by area under curve spectrophotometric method. The study utilized methanol as a solvent and area between the wavelengths of 241nm-260nm was

measured.

A study on new spectrophotometric method for the quantification of rivaroxaban was reported by Sekaran C.B., et al (2013)30 conducted. In this method rivaroxaban in dimethyl sulphoxide was estimated at 270nm. The linearity

(27)

Satyanarayana P.V.V., et al (2013)31 reported a spectrophotometric technique for the estimation of rivaroxaban. The method utilized principle of the color formation of the rivaroxaban on binding with different reagents. The method reported linearity ranges of 2 to 90 ppm of rivaroxaban estimated using five

different reagents.

A RP-HPLC method for estimation of rivaroxaban was established by

Mustafa C., et al (2013)32. The isocratic elution was performed with ACN: water

in a ration of 55:45 v/v and the detection was done at 249nm. The rivaroxaban

was eluted at retention time of 3.37 min and the linearity was reported in concentration range of 5- 40 µg/ml.

Satyanarayana P.V.V., et al (2012)33 reported RP-HPLC method for the

quatification of rivaroxaban. The mobile phase of THF:methanol:ACN was employed in ratios of 10:40:50. The scanning was at 270 nm and the reported Rt of rivaroxaban was 2.55min. The reported linearity concentration ranges are 40

-100 µg/ml.

A novel HPLC method for estimation of rivaroxaban described by

Nallagatla V.B.R., et al (2013)34. The mobile phase consisted of Methanol: ACN

was employed. The retention time of rivaroxaban was reported at 5.35 min and linearity was reported between 10-100 µg/ml.

Pinaz A.K., (2013)35 developed RP-HPLC method for conducting

phytolytic-thermal degradation of rivaroxaban. An isocratic separation was conducted with mobile phase consisted of 50:50 v/v methanol: acetonitrile. PDA detector was used and the detection wavelength of 250 nm was fixed. The linearity was found between the range of 20-100 µg/ml.

Darshna V., et al (2014)36 has reported HPTLC method for estimation of

(28)

3.4. Ticagrelor

A spectrophotometric method was reported by Mrunal A.A., et al

(2014)37 to determine ticagrelor. Methanol was selected as a solvent and the

absorption maximum of ticagrelor was reported at 255nm. Linearity was obeyed in range of 2-7μg/mL. The recovery of ticagrelor ranged from 99.59% to 101.66%.

Lakshmana R., et al (2013)38 established HPLC method to determine

ticagrelor. The mobile phase composed of phosphate buffer and ACN was used along with the C18 column as a stationary phase. The method described linear response between 22.5-135 μg/mL. The percentage assay of ticagrelor was found to be 99.9%.

(29)

4. SCOPE & PLAN OF THE STUDY

SCOPE:

An extensive literature survey was carried out and various analytical methods reported were analyzed. There are some reported UV spectroscopy19-21, HPLC22-26 and HPTLC27 methods for the estimation of dronedarone hydrochloride. There are no reported methods for determination of Levosimendan.

There are also some reported UV spectroscopic methods29-31, HPLC32-35 and HPTLC36 for the quantification of rivaroxaban in its formulation. The UV spectrophotometric method37and validated stability indicating HPLC38 method was also reported for the determination of ticagrelor. Quantification of ticagrelor in human plasma using ultra-performance liquid chromatography was also

reported39. The reported analytical methods for the selected drugs involve usage of mobile phase systems which are costlier or less sensitive or tedious. There is a need for simple, sensitive and economical analytical methods for the determination of selected drugs. Hence the newer analytical method development for the selected drug will be useful in assay of their formulation and in-vitro drug

interaction studies with co-administered drugs.

PLAN OF STUDY:

Phase I: Development of analytical methods for the assay of selected

drugs.

UV-Visible method - It involves various parameters like selection of

solvent, fixing of wavelength, Beer’s law concentration showing linearity and range and application to formulations.

Fluorimetric method - It involves selection of fluoro tags for various

(30)

HPTLCmethod- The steps involved in method development are selection of pre-coated chromatographic plate, selection of solvent system, application of sample, detection of spots, scanning and documentation.

HPLC method - It involves the fixing up of experimental conditions such

as selection of solvent type, column type, mobile phase, effects of ionic strength, selectivity like solvent strength and temperature. It also involves ensuring limits of

parameters for system suitability.

Phase II: Validation of the developed method- After development of the

method, it has been validated as per guidelines for validation. Accuracy can be studied by recovery studies. Precision can be studied by evaluation of repeatability of the method.

Phase III: Application of the methods for the assay of formulations.

Phase IV: Application of the developed validated methods for the stability

indicating studies and in-vitro drug interaction studies.

Stability indicating studies: Forced degradation study by applying the

developed HPLC or HPTLC technique. The drugs will be subjected to acid, base and neutral hydrolysis, hydrogen peroxide induced oxidation and photochemical degradation. The analyte as well as degradation products may be resolved using validated RP-HPLC or HPTLC techniques.

In-vitro displacement interaction study: The in-vitro protein binding of

drugs are measured by equilibrium dialysis method using plasma proteins. Quantification of the free drug may be carried out using the newly developed and validated analytical technique. The fraction of unbound drug is then determined

after dialysis. The percentage displacement of study drug from protein in presence of interacting drug will be estimated after equilibrium dialysis of both drugs with protein.

Statistical Analysis: The data generated are analyzed through suitable

(31)

5. MATERIALS AND INSTRUMENTS

MATERIALS:

Reference standards used in the study were having HPLC purity >99% of dronedarone hydrochloride, rivaroxaban, levosimendan, ticagrelor, aspirin,

clopidogrel and atorvastatin were purchased from reputed suppliers with certificate of analysis. HPLC grade solvents like methanol, dimethyl sulphoxide and acetonitrile were supplied by S.D. Fine Chemicals. Rest of the chemicals and reagents used weresupplied byRanbaxy chemicals. Dialysis membrane tubes and

bovine serum albumin (BSA) obtained from Hi-Media lab. Formulations containing the selected cardiovascular drugs were procured from pharmacy.

INSTRUMENTS:

 Jasco V-630 UV/VIS spectrophotometer

 JASCO FP -750 spectrofluorimeter

 Linomat 5 sampler applicator equipped with a 100µL syringe

 Twin-trough glass chamber (CAMAG)

 CAMAG TLC scanner 3

 A Shimadzu LCMS – 2010EV system

 Shimadzu digital Electronic Balance BL – 220 H

 Systronic digital pH meter MK (VI)

 Orbital shaker supplied by Sub zero lab instruments

 Leelasonic ultrasonic sonicator

(32)

DRUG PROFILE

Generic Name : Dronedarone Hydrochloride40-42

Chemical Structure :

Molecular formula : C31H44N2O5S.HCl Molecular Weight : 593.22 g/mol

IUPAC Name : N-{2-butyl-3-[4-(3-dibutylaminopropoxy) benzoyl]benzofuran-5-yl}methanesulfonamide

hydrochloride Appearance : Fine white powder Physical state : Solid

Solubility : Soluble in methanol and Methylene Chloride. Category : Anti-arrhythmic agent

CDSCO approval date : 08/04/2010

Protein Binding : > 98 % binds mainly to albumin

Marketed Formulations:

Brand Name Formulation Strength

(33)

Generic Name : Levosimendan43-45

Molecular formula : C31H44N2O5S.HCl

Molecular Weight : 280.28 g/mol

IUPAC Name : 2-[[4-[(4R)-4-methyl-6-oxo-4,5-dihydro-1H- pyridazin-3-yl] phenyl] hydrazinylidene] propanedinitrile

Appearance : Crystalline yellow powder Physical state : Solid

Solubility : Completely soluble in methanol. Category : Calcium sensitizer used in CHF CDSCO approval date : 30/04/2010

Protein Binding : 98% bound to plasma protein

Marketed Formulations:

(34)

Generic Name : Rivaroxaban46-48

Molecular formula : C28H36N4O2S Molecular Weight : 492.676 g/mol

IUPAC Name : 5-chloro-N-{[(5S)-2-oxo-3-[4-(3-oxomorpholin- 4-yl)phenyl]-1,3- oxazolidin-5- yl] methyl}

thiophene-2- carboxamide

Appearance : White to off-white crystalline powder Physical state : Solid

Solubility : Soluble in DMSO and is practically insoluble in water and aqueous media.

Category : Anticoagulant drug

CDSCO approval date :

30.10.2010

Protein Binding : > 95 % binds mainly to albumin Marketed Formulations:

(35)

Generic Name : Ticagrelor49-50

Molecular formula : C23H28F2N6O4S Molecular Weight : 522.56 g/mol

IUPAC Name : (1S,2S,3R,5S)-3-(7-{[(1R,2S)-2-(3,4- difluorophenyl)cyclopropyl]amino}-5-(propylsulfanyl)-3H -[1,2,3]triazolo[4,5-d]

pyrimidin-3-yl)-5-(2- hydroxyethoxy) cyclopentane-1,2-diol

Appearance : Fine white powder Physical state : Solid

Solubility : Freely soluble in methanol. Category : Platelet aggregation inhibitor

CDSCO approval date : 03.05.2012

Protein Binding : > 99 % to plasma proteins Marketed Formulations:

(36)

6. EXPERIMENTAL METHODS

6.1.1. DEVELOPMENT OF VALIDATED UV SPECTROPHOTOMETRIC

METHOD FOR THE DETERMINATION OF DRONEDARONE

HYDROCHLORIDE IN TABLET DOSAGE FORM

6.1.1.1 Method development

The solvent selection was done by assessing the solubility of dronedarone

hydrochloride in different solvents. It was found that drug was completely soluble in methanol. Appropriate concentration of dronedarone hydrochloride in methanol was scanned to record the spectra. The normal spectrum was recorded using the standard solutions and converted to various derivative spectra, from which second order derivative spectrum was selected. The band at which highest absorption was observed is selected for analysis.

6.1.1.2 Preparation of standard solutions

Exactly weighed 10mg of dronedarone hydrochloride was taken and

dissolved in methanol. The volume was made up to 10ml. Further dilutions were done with methanol to achieve required concentrations.

6.1.1.3 Method validation

The validation was done as per ICH guidelines Q2(R1). The validation parameters studied are as follows;

Linearity: The working standard solutions of dronedarone hydrochloride

were scanned against blank and the absorbance was recorded at the λmax of 292 nm. The normal spectra obtained were converted to second order derivative spectra using the software. Calibration graph was obtained by plotting concentration against absorbance.

Specificity: Placebo was prepared51 with mixture of common excipients

(37)

Accuracy: A known concentration of reference was added to a pre-analyzed sample solution. The reference was added and recovery was calculated. The ratio of difference between total concentration of the analyte found and the concentration of the analyte present in pre-analyzed sample.

Precision: The precision was demostrated by repeatability (intra-day) and

intermediate precision (inter-day) studies. The intra-day precision studies were done by repeating measurements on the same day. Inter-day precision was carried out by measuring absorbance of three different concentrations on different days.

LOD and LOQ: It is calculated by the equation: LOD = 3.3(σ /S) and

LOQ = 10(σ /S), where, σ = Standard deviation of regression line and S = Slope of

the calibration curve.

Stability of solution: The stock solutions stored under room temperature

and refrigerated conditions (8°C±0.5°C) were subjected to stability study. The solutions were diluted to suitable concentration and the absorbance was measured at different time intervals.

6.1.1.4 Application of the UV spectrophotometric method for the assay of

dronedarone hydrochloride in tablet dosage form

The assay of dronedarone hydrochloride tablets was carried out by the validated second order derivative UV spectrophotometric method. Twenty tablets

of marketed formulation were weighed and powdered. The amount of powder equivalent to 10mg of dronedarone hydrochloride was taken and it was dissolved

in methanol. The solution was sonicated for 20 minutes and the volume was made up with methanol. A suitable aliquot of the filtered solution was diluted to achieve the required concentration. The absorbance of diluted solution was measured against blank at 292nm. The zero order spectrum recorded from the formulation, which was derivatized to obtain second order spectrum. The absorbance was used

(38)

6.1.2. DEVELOPMENT OF VALIDATED SPECTROFLUORIMETRIC

METHOD FOR THE DETERMINATION OF DRONEDARONE

6.1.2.1 Method development

The method was based on the oxidation of dronedarone hydrochloride with CAS. The fluorescence intensity produced by cerium (III) was measured. The selection of solvent was done based on solubility of drug and methanol was selected to prepare stock solutions. Preparation of working standard was done based on trials with different solvents and water was selected. Oxidation of drug using 0.0005M

cerric ammonium sulphate in 0.05M sulphuric acid has been carried out by heating the mixture at 50 ºC using water bath for 20minutes. The spectrofluorimetric analysis was performed by scanning the oxidized samples in the region of 300-600 nm. The spectrum was recorded and fluorescence intensity were noted.

6.1.2.2 Preparation of reaction solutions

Preparation of cerric ammonium sulphate (CAS) solution: To prepare

0.0005M CAS solution, 0.0316 gm of CAS was dissolved in 0.05M sulphuric acid. The volume was made up to 100ml with 0.05M sulphuric acid.

Preparation standard solutions: From standard dronedarone hydrochloride

10mg was taken and dissolved in methanol. The volume was made up to 10ml with methanol. Working standard solution was prepared by appropriately diluting the stock

with water.

Preparation of reaction mixture: To the 2ml of CAS solution in 10ml

volumetric flasks, different volumes (1-10ml) of appropriately diluted drug solution

(2µg/ml) was added and diluted up to 10ml with water. The solution was heated at 40ºC for 30 minutes and cooled to room temperature.

6.1.2.3. Optimization of reaction condition

Acid and its concentration: Various acid like hydrochloric acid, nitric acid

(39)

Concentration and volume of CAS: Different concentrations (0.00025-0.001M) of cerric ammonium sulphate were used to evaluate its effect on fluorescence intensity. The effect of different volume of CAS solution (1-5ml) used for the reaction on fluorescence intensity was determined.

Effect of reaction temperature: The effect of reaction temperature on

fluorescence intensity of cerium (III) was evaluated by applying different temperatures (40 ºC -60 ºC) to reaction mixture.

Effect of heating time: The effect of heating time (10-50 min) on reaction mixture was studied. The optimum heating time that produces maximum intensity was

selected and applied for further studies.

Instrumental parameters: The band width and response time have been

varied to study their effect on fluorescence intensity.

6.1.2.4. Optimized conditions

The required volume of drug solution taken, 2ml of 0.0005M CAS solution on 0.05M suphuric acid was added to the volumetric flask containing drug solution. The solution was diluted to 10ml using water. and the flask was heated for 30 minutes in a water bath at 50 ºC. The mixture was further cooled and the intensity of cerium (III) was recorded. at prefixed excitation (λex=278 nm) and emission (λem=364nm)

wavelengths

6.1.2.5. Method validation

The developed spectofluorimetric method for determination of dronedarone

hydrochloride is further validated as per ICH guidelines.

Linearity: The fluorescence intensity of oxidized dronedarone hydrochloride

solutions were recorded against blank. Calibration graph plotted and the linearity concentrations were determined.

Specificity: The spectra obtained from placebo and assay sample were

compared with standard for any change in the spectra due to the presence of

(40)

Accuracy: A known concentration of reference was added to a pre-analyzed sample solution of dronedarone hydrochloride. The percentage recovery and %RSD value were calculated.

Precision: It wasevaluated by intra-day and inter-day studies. The intra-day precision studies were done by measuring fluorescence intensity three times for three different concentrations with in a day. Inter-day precision was carried out by measuring fluorescence intensity on three different days.

LOD and LOQ: On the basis of slope value obtained, LOD and LOQ were

determined from equation.

Stability of stocks: The stock solutions kept in bench top and refrigerated conditions (8°C±0.5°C) were subjected to the reaction after appropriate dilutions. The fluorescence intensity of the resulting reaction mixtures was recorded. The stability of the solution at different time intervals was determined by calculating the percentage

amount of drug remaining in the solution.

Instrumental parameters: The effect of band width on the fluorescence

intensity were studied by changing the band width 5,5 and 5,10 and also scanning speed was varied to different levels to check its effect on the fluorescence

measurement.

6.1.2.4 Application of the spectrofluorimetric method for the assay of

The assay of dronedarone hydrochloride tablets was carried out by the

validated spectrofluorimetric method. From the powdered tablets quantity equivalent to 10mg of drug was taken and it was dissolved in methanol. The solution was

(41)

6.1.3. DEVELOPMENT OF VALIDATED HPTLC METHOD FOR THE

DETERMINATION OF DRONEDARONE HYDROCHLORIDE IN TABLET

DOSAGE FORM

6.1.3.1. Method development

Based on the solubility of drug in different solvents, trials were done with acetonitrile, methanol and chloroform. It was found that dronedarone had better solubility in methanol and it was selected for the preparation of stock. Samples were spotted as bands using sampler applicator on a 10 × 10 cm silica gel G60F254 plate which was selected as a stationary phase. Linear ascending development was performed and scanning of plates was performed. It was found that at 292 nm dronedarone hydrochloride had a maximum absorbance and thus λmax of 292 nm was selected for further analysis.

Selection of mobile phase: The various composition of mobile phase tried

includes methanol: toluene, methanol: choloroform, methanol: n-hexane and methanol: water. Among all methanol: water resulted in good separation with compact spots and it was selected as a mobile phase for further study.

6.1.3.2. Optimization of chromatographic condition

Ratio of mobile phase: Effect of different ratios of methanol : water was studied tried, from which the ratio of (9:1v/v) was selected because it gave good separation with compact spots. The peaks are symmetric and the Rf value was found to be 0.8.

Optimization of chamber saturation: The above fixed mobile phase ratio

was taken in development chamber and different saturation time from 5 to 30 minutes were tried. 15 minutes of chamber saturation time was fixed for the method.

6.1.3.3 Fixed experimental conditions

Stationary phase : Pre-coated silica gel G60F254 plates Mobile phase : Methanol : water (9:1 v/v)

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Band width : 6mm

Source of radiation : Deuterium lamp

6.1.3.4 Preparation standard solutions

From standard dronedarone hydrochloride 10mg was taken and dissolved using methanol. The volume was made up to 10ml. Further dilution of stock was done to get desired concentration.

6.1.3.5 Method validation

The validation was carried out by assessing linearity, repeatability, inter and intra-day precision, LOD, LOQ, plate stability and solution stability.

Linearity and Range:From 10μg/ml solution of dronedarone hydrochloride

1-10 μl was applied on a 10 ×10 TLC plate with Linomat 5 applicator. The plate was developed using Methanol : water (9:1v/v). The plate was scanned and peak areas were measured at 292nm.

Accuracy: It was determined by calculating recovery of Dronedarone

hydrochloride. The recovery of Dronedarone hydrochloride was estimated by comparing the peak area of standards. Calculation of percentage recovery and %RSD were done.

Repeatability: Repeatability was analyzed by repeating sample application

and sample measurement. Spotting a particular volume in linearity range was repeated six times and the repeatability was ensured. The peak areas were analyzed and % RSD was calculated. Repeatability of measurement was also performed by scanning of plate for six times and the %RSD value of result was calculated.

Intra-day & Inter-day precision: Intra-day precision was studied by spotting

three different volumes on the same day. Inter day precision was studied by performing the method on different days and % RSD was calculated.

LOD and LOQ: On the basis of slope obtained LOD and LOQ were

calculated using standard equation.

(43)

Stability of Solution: The stability of stock solutions stored under normal and refrigerated conditions (8°C±0.5°C) were analyzed. The solutions were diluted to suitable concentration for spotting. The required volume was spotted at various time intervals, plates were developed and scanned.

6.1.3.6. Application of the developed HPTLC method for the assay of

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6.1.4. DEVELOPMENT OF VALIDATED STABILITY INDICATING

RP-HPLC METHOD FOR THE DETERMINATION OF DRONEDARONE

6.1.4.1. Method development

Selection of stationary phase: Since dronedarone hydrochloride is polar in

nature reversed phase method was selected for the study. Based on molecular weight and solubility of dronedarone hydrochloride RP-HPLC method with C18 column was employed for the method development.

Selection of wavelength: The wavelength selection was done with the help of

UV spectrum of dronedarone hydrochloride. An UV spectrum of dronedarone hydrochloride was recorded and it was found that the drug has maximum absorbance at the wavelength of 292 nm. It was fixed for the detection of dronedarone hydrochloride.

Selection of mobile phase: Trials were done with different buffers and ACN

in various ratios. The disodium hydrogen phosphate buffer and ACN resulted in peak with good shape and hence it was selected for further analysis.

6.1.4.2. Initial conditions

A Hibar®RP – C18 HPLC column was selected as a stationary phase. The selected column has the advantage of high degree of retention, better reproducibility and ability to produce lower back pressure. The mobile phase selected were disodium hydrogen phosphate buffer and ACN in a ratio of 50:50%v/v. The detection was carried out at 292 nm and separation was achieved in room temperature.

Stock solution preparation: From standard drug 10mg was taken and it was

dissolved with methanol. The volume was made up to 10ml. Further dilutions were done with methanol.

Working standard preparation: From dronedarone hydrochloride stock, 1ml

was taken and the volume was made up to 10ml with methanol. Further serial dilutions were made to get required concentration ranges.

Preparation of buffer: To prepare 0.01M of disodium hydrogen phosphate